Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 15 de 15
Filtrar
1.
J Biol Chem ; 299(1): 102792, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36516886

RESUMO

The GTPase-activating protein (GAP) p190RhoGAP (p190A) is encoded by ARHGAP35 which is found mutated in cancers. p190A is a negative regulator of the GTPase RhoA in cells and must be targeted to RhoA-dependent actin-based structures to fulfill its roles. We previously identified a functional region of p190A called the PLS (protrusion localization sequence) required for localization of p190A to lamellipodia but also for regulating the GAP activity of p190A. Additional effects of the PLS region on p190A localization and activity need further characterization. Here, we demonstrated that the PLS is required to target p190A to invadosomes. Cellular expression of a p190A construct devoid of the PLS (p190AΔPLS) favored RhoA inactivation in a stronger manner than WT p190A, suggesting that the PLS is an autoinhibitory domain of p190A GAP activity. To decipher this mechanism, we searched for PLS-interacting proteins using a two-hybrid screen. We found that the PLS can interact with p190A itself. Coimmunoprecipitation experiments demonstrated that the PLS interacts with a region in close proximity to the GAP domain. Furthermore, we demonstrated that this interaction is abolished if the PLS harbors cancer-associated mutations: the S866F point mutation and the Δ865-870 deletion. Our results are in favor of defining PLS as an inhibitory domain responsible for masking the p190A functional GAP domain. Thus, p190A could exist in cells under two forms: an inactive closed conformation with a masked GAP domain and an open conformation allowing p190A GAP function. Altogether, our data unveil a new mechanism of p190A regulation.


Assuntos
Fatores de Troca do Nucleotídeo Guanina , Neoplasias , Humanos , Actinas/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Mutação , Mutação Puntual , Pseudópodes/metabolismo , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Domínios Proteicos
2.
Exp Cell Res ; 370(2): 227-236, 2018 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-29940177

RESUMO

Rnd3/RhoE is an atypical member of the Rho family of small GTPases, devoid of intrinsic GTP hydrolytic activity and a general modulator of important cellular processes such as migration and proliferation. Here, we show that Rnd3 is a target of the transcription factor SRF and its co-activator MKL1. The MKL1-SRF pathway assures the translation of physical forces into a transcriptional response. Rho GTPases can modulate the activity of this mechanotransduction pathway through actin cytoskeleton regulation, and many MKL1-SRF targets are involved in the regulation of actin. We found that Rnd3 expression is altered by G-actin signaling and sensitive to actin-targeting drugs and MKL1 mutants. We further characterized a consensus SRF binding site in the Rnd3 promoter. We found that MKL1-SRF modulation regulates Rnd3 promoter activity and Rnd3 expression can affect MKL1-SRF pathway activity in return. We demonstrated that this novel MKL1-SRF target is required in mechanosensitive mechanisms such as cell spreading and spheroid formation. Thus, Rnd3 is a MKL1-SRF target that plays a key role in the feedback loop described between the MKL1-SRF pathway and the organization of the actin cytoskeleton.


Assuntos
Actinas/metabolismo , Transativadores/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Citoesqueleto de Actina/metabolismo , Células Cultivadas , Humanos , Mecanotransdução Celular/fisiologia , Regiões Promotoras Genéticas/genética , Fator de Resposta Sérica/metabolismo
3.
Cell Biochem Funct ; 35(6): 289-295, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28833338

RESUMO

Hepatocellular carcinoma (HCC) is the main primary cancer of the liver. Many studies have shown that insulin resistance is a risk factor for HCC. We previously discovered the overexpression and oncogenic role of the Reptin/RUVBL2 ATPase in HCC. Here, we found that Reptin silencing enhanced insulin sensitivity in 2 HCC cell lines, as shown by a large potentiation of insulin-induced AKT phosphorylation on Ser473 and Thr308, and of downstream signalling. Reptin silencing did not affect the tyrosine phosphorylation of the insulin receptor nor of IRS1, but it enhanced the tyrosine phosphorylation of the p85 subunit of PI3K. The expression of the SHP-1/PTPN6 phosphatase, which dephosphorylates p85, was reduced after Reptin depletion. Forced expression of SHP-1 restored a normal AKT phosphorylation after insulin treatment in cells where Reptin was silenced, demonstrating that the downregulation of SHP1 is mechanistically linked to increased Akt phosphorylation. In conclusion, we have uncovered a new function for Reptin in regulating insulin signalling in HCC cells via the regulation of SHP-1 expression. We suggest that the regulation of insulin sensitivity by Reptin contributes to its oncogenic action in the liver.


Assuntos
Proteínas de Transporte/metabolismo , DNA Helicases/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , ATPases Associadas a Diversas Atividades Celulares , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/genética , Linhagem Celular Tumoral , DNA Helicases/antagonistas & inibidores , DNA Helicases/genética , Doxiciclina/farmacologia , Humanos , Insulina/farmacologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Fosforilação/efeitos dos fármacos , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/efeitos dos fármacos
4.
JHEP Rep ; 5(5): 100691, 2023 May.
Artigo em Inglês | MEDLINE | ID: mdl-37153687

RESUMO

Background & Aims: ß-catenin is a well-known effector of the Wnt pathway, and a key player in cadherin-mediated cell adhesion. Oncogenic mutations of ß-catenin are very frequent in paediatric liver primary tumours. Those mutations are mostly heterozygous, which allows the co-expression of wild-type (WT) and mutated ß-catenins in tumour cells. We investigated the interplay between WT and mutated ß-catenins in liver tumour cells, and searched for new actors of the ß-catenin pathway. Methods: Using an RNAi strategy in ß-catenin-mutated hepatoblastoma (HB) cells, we dissociated the structural and transcriptional activities of ß-catenin, which are carried mainly by WT and mutated proteins, respectively. Their impact was characterised using transcriptomic and functional analyses. We studied mice that develop liver tumours upon activation of ß-catenin in hepatocytes (APCKO and ß-cateninΔexon3 mice). We used transcriptomic data from mouse and human HB specimens, and used immunohistochemistry to analyse samples. Results: We highlighted an antagonistic role of WT and mutated ß-catenins with regard to hepatocyte differentiation, as attested by alterations in the expression of hepatocyte markers and the formation of bile canaliculi. We characterised fascin-1 as a transcriptional target of mutated ß-catenin involved in tumour cell differentiation. Using mouse models, we found that fascin-1 is highly expressed in undifferentiated tumours. Finally, we found that fascin-1 is a specific marker of primitive cells including embryonal and blastemal cells in human HBs. Conclusions: Fascin-1 expression is linked to a loss of differentiation and polarity of hepatocytes. We present fascin-1 as a previously unrecognised factor in the modulation of hepatocyte differentiation associated with ß-catenin pathway alteration in the liver, and as a new potential target in HB. Impact and implications: The FSCN1 gene, encoding fascin-1, was reported to be a metastasis-related gene in various cancers. Herein, we uncover its expression in poor-prognosis hepatoblastomas, a paediatric liver cancer. We show that fascin-1 expression is driven by the mutated beta-catenin in liver tumour cells. We provide new insights on the impact of fascin-1 expression on tumour cell differentiation. We highlight fascin-1 as a marker of immature cells in mouse and human hepatoblastomas.

5.
World J Gastroenterol ; 14(14): 2194-9, 2008 Apr 14.
Artigo em Inglês | MEDLINE | ID: mdl-18407593

RESUMO

AIM: To test the effect of a standardized red wine polyphenolic extract (RWPE) on the phenotype of human liver myofibroblasts in culture. METHODS: Human myofibroblasts grown from liver explants were used in this study. Cell proliferation was measured with the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay. Signaling events were analyzed by western blot with phospho-specific antibodies. Matrix-metalloproteinase activity was measured with gel zymography. RESULTS: We found that cell proliferation was dose-dependently decreased by up to 90% by RWPE while cell viability was not affected. Exposure to RWPE also greatly decreased the phosphorylation of ERK1/ERK2 and Akt in response to stimulation by the mitogenic factor platelet-derived growth factor BB (PDGF-BB). Finally, RWPE affected extracellular matrix remodeling by decreasing the secretion by myofibroblasts of matrix-metalloproteinase-2 and of tissue inhibitor of matrix-metalloproteinases-1. CONCLUSION: Altogether, RWPE decreases the activation state of liver myofibroblasts. The identification of the active compounds in RWPE could offer new therapeutic strategies against liver fibrosis.


Assuntos
Fibroblastos/metabolismo , Flavonoides/farmacologia , Fígado/patologia , Miofibrilas/metabolismo , Fenóis/farmacologia , Vinho , Actinas/metabolismo , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , DNA/metabolismo , Fibrose , Flavonoides/química , Humanos , Fígado/metabolismo , Fenóis/química , Fenótipo , Fosforilação , Polifenóis , Sais de Tetrazólio/farmacologia , Tiazóis/farmacologia
6.
Gastroenterol Clin Biol ; 31(2): 141-5, 2007 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17347620

RESUMO

AIMS: The aim of this study was to evaluate the serum pattern of cytokines evolution after surgical radiofrequency ablation (SRFA) of colorectal metastases. METHODS: Metastases of ten non consecutive patients were destroyed by radiofrequency ablation without concomitant resection after a complete surgical procedure including a laparotomy, a peritoneal examination, liver mobilisation and liver ultrasound. Serum levels of IL-6, TNFalpha, HGF, VEGF, bFGF, TGFbeta1 and CRP were assessed by ELISA assays at different time points. RESULTS: TNFalpha and bFGF remained undetectable. IL-6 peaked at 3 hours and remained elevated during the entire study period. HGF increased by three-fold by Day 1 then decreased until Day 7 where it was still twice its baseline level. VEGF level increased from Day 5 onward. TGFbeta1 did not show significant variations. CRP was increased throughout the study. CONCLUSIONS: In contrast with cryotherapy, SRFA does not lead to high serum TNFalpha suggesting a better tolerance. Nevertheless high IL-6, HGF and VEGF serum levels are characteristic of a general inflammatory stress which should be taken into account.


Assuntos
Ablação por Cateter , Neoplasias Colorretais/patologia , Citocinas/sangue , Neoplasias Hepáticas/cirurgia , Idoso , Humanos , Neoplasias Hepáticas/secundário , Pessoa de Meia-Idade
7.
Oncotarget ; 8(5): 7839-7851, 2017 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-27999200

RESUMO

Hepatocellular carcinoma is associated with a high rate of intra-hepatic invasion that carries a poor prognosis. Meprin alpha (Mep1A) is a secreted metalloproteinase with many substrates relevant to cancer invasion. We found that Mep1A was a target of Reptin, a protein that is oncogenic in HCC. We studied Mep1A regulation by Reptin, its role in HCC, and whether it mediates Reptin oncogenic effects.MepA and Reptin expression was measured in human HCC by qRT-PCR and in cultured cells by PCR, western blot and enzymatic activity measurements. Cell growth was assessed by counting and MTS assay. Cell migration was measured in Boyden chambers and wound healing assays, and cell invasion in Boyden chambers.Silencing Reptin decreased Mep1A expression and activity, without affecting meprin ß. Mep1A, but not meprin ß, was overexpressed in a series of 242 human HCC (2.04 fold, p < 0.0001), and a high expression correlated with a poor prognosis. Mep1A and Reptin expressions were positively correlated (r = 0.39, p < 0.0001). Silencing Mep1A had little effect on cell proliferation, but decreased cell migration and invasion of HuH7 and Hep3B cells. Conversely, overexpression of Mep1A or addition of recombinant Mep1A increased migration and invasion. Finally, overexpression of Mep1A restored a normal cell migration in cells where Reptin was depleted.Mep1A is overexpressed in most HCC and induces HCC cell migration and invasion. Mep1A expression is regulated by Reptin, and Mep1A mediates Reptin-induced migration. Overall, we suggest that Mep1A may be a useful target in HCC.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Carcinoma Hepatocelular/enzimologia , Proteínas de Transporte/metabolismo , Movimento Celular , DNA Helicases/metabolismo , Neoplasias Hepáticas/enzimologia , Metaloendopeptidases/metabolismo , ATPases Associadas a Diversas Atividades Celulares/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Proliferação de Células , DNA Helicases/genética , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Metaloendopeptidases/genética , Invasividade Neoplásica , Interferência de RNA , Transdução de Sinais , Fatores de Tempo , Transfecção
8.
FEBS J ; 273(5): 1056-64, 2006 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-16478478

RESUMO

Dipeptidyl-peptidase III (DPP III) hydrolyses small peptides with a broad substrate specificity. It is thought to be involved in a major degradation pathway of the insect neuropeptide proctolin. We report the purification and characterization of a soluble DPP III from 40 g Drosophila melanogaster. Western blot analysis with anti-(DPP III) serum revealed the purification of two proteins of molecular mass 89 and 82 kDa. MS/MS analysis of these proteins resulted in the sequencing of 45 and 41 peptide fragments, respectively, confirming approximately 60% of both annotated D. melanogaster DPP III isoforms (CG7415-PC and CG7415-PB) predicted at 89 and 82 kDa. Sequencing also revealed the specific catalytic domain HELLGH in both isoforms, indicating that they are both effective in degrading small peptides. In addition, with a probe specific for D. melanogaster DPP III, northern blot analysis of fruit fly total RNA showed two transcripts at approximately 2.6 and 2.3 kb, consistent with the translation of 89-kDa and 82-kDa DPP III proteins. Moreover, the purified enzyme hydrolyzed the insect neuropeptide proctolin (Km approximately 4 microm) at the second N-terminal peptide bound, and was inhibited by the specific DPP III inhibitor tynorphin. Finally, anti-(DPP III) immunoreactivity was observed in the central nervous system of D. melanogaster larva, supporting a functional role for DPP III in proctolin degradation. This study shows that DPP III is in actuality synthesized in D. melanogaster as 89-kDa and 82-kDa isoforms, representing two native proteins translated from two alternative mRNA transcripts.


Assuntos
Dipeptidil Peptidases e Tripeptidil Peptidases/isolamento & purificação , Drosophila melanogaster/enzimologia , Animais , Sequência de Bases , Sistema Nervoso Central/enzimologia , DNA/genética , Dipeptidil Peptidases e Tripeptidil Peptidases/química , Dipeptidil Peptidases e Tripeptidil Peptidases/genética , Dipeptidil Peptidases e Tripeptidil Peptidases/metabolismo , Imuno-Histoquímica , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/isolamento & purificação , Isoenzimas/metabolismo , Cinética , Espectrometria de Massas , Peso Molecular , Solubilidade
9.
PLoS One ; 10(4): e0123333, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25875766

RESUMO

Reptin/RUVBL2 is overexpressed in most hepatocellular carcinomas and is required for the growth and viability of HCC cells. Reptin is involved in several chromatin remodeling complexes, some of which are involved in the detection and repair of DNA damage, but data on Reptin involvement in the repair of DNA damage are scarce and contradictory. Our objective was to study the effects of Reptin silencing on the repair of DNA double-strand breaks (DSB) in HCC cells. Treatment of HuH7 cells with etoposide (25 µM, 30 min) or γ irradiation (4 Gy) increased the phosphorylation of H2AX by 1.94 ± 0.13 and 2.0 ± 0.02 fold, respectively. These values were significantly reduced by 35 and 65 % after Reptin silencing with inducible shRNA. Irradiation increased the number of BRCA1 (3-fold) and 53BP1 foci (7.5 fold). Depletion of Reptin reduced these values by 62 and 48%, respectively. These defects in activation and/or recruitment of repair proteins were not due to a decreased number of DSBs as measured by the COMET assay. All these results were confirmed in the Hep3B cell line. Protein expression of ATM and DNA-PKcs, the major H2AX kinases, was significantly reduced by 52 and 61 % after Reptin depletion whereas their mRNA level remained unchanged. Phosphorylation of Chk2, another ATM target, was not significantly altered. Using co-immunoprecipitation, we showed an interaction between Reptin and DNA-PKcs. The half-life of newly-synthesized DNA-PKcs was reduced when Reptin was silenced. Finally, depletion of Reptin was synergistic with etoposide or γ irradiation to reduce cell growth and colony formation. In conclusion, Reptin is an important cofactor for the repair of DSBs. Our data, combined with those of the literature suggests that it operates at least in part by regulating the expression of DNA-PKcs by a stabilization mechanism. Overexpression of Reptin in HCC could be a factor of resistance to treatment, consistent with the observed overexpression of Reptin in subgroups of chemo-resistant breast and ovarian cancers.


Assuntos
Proteínas de Transporte/genética , Quebras de DNA de Cadeia Dupla , DNA Helicases/genética , Reparo do DNA/genética , Interferência de RNA , ATPases Associadas a Diversas Atividades Celulares , Antineoplásicos Fitogênicos/farmacologia , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Western Blotting , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Proliferação de Células/efeitos da radiação , Ensaio Cometa , DNA Helicases/metabolismo , Proteína Quinase Ativada por DNA/genética , Proteína Quinase Ativada por DNA/metabolismo , Etoposídeo/farmacologia , Raios gama , Histonas/metabolismo , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Microscopia Confocal , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilação/efeitos dos fármacos , Fosforilação/efeitos da radiação , Ligação Proteica , Reação em Cadeia da Polimerase Via Transcriptase Reversa
10.
Thromb Haemost ; 91(3): 569-75, 2004 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-14983234

RESUMO

Tissue factor pathway inhibitor-2 (TFPI-2) is a recently described serine proteinase inhibitor. Human and murine TFPI-2 share about 50% homology. The aim of this study was to investigate the cellular localization of human and murine TFPI-2 in the liver and the regulation of their expression during acute inflammation. Northern blot, in situ hybridization and studies on isolated hepatocytes demonstrated a high-level expression of TFPI-2 in murine hepatocytes. On the other hand, very little TFPI-2 mRNA expression could be detected in human liver. Studies with isolated human liver cells suggested that TFPI-2 expression in human liver was mainly observed in liver sinusoidal endothelial cells rather than hepatocytes. Liver murine TFPI-2 expression was greatly increased after lipopolysaccharide administration with a delayed kinetics as compared to alpha1-acid glycoprotein, a classical acute-phase reactant. Accordingly, studies with isolated cells showed that the increase in TFPI-2 transcripts occurred in non-hepatocytic cells. Moreover, the LPS response was abolished in mice with a hepatocyte-specific KO for the gp130 receptor, thus indicating that a mediator from hepatocytes is involved in the up-regulation of TFPI-2 in non-parenchymal cells. In conclusion, murine TFPI-2 is highly expressed in hepatocytes in the normal murine liver and is upregulated in non-parenchymal cells in the context of inflammation. The large difference in the level of liver expression of human and murine TFPI-2 suggests that despite significant sequence similarities, these proteins presumably have different functions in the two species.


Assuntos
Glicoproteínas/biossíntese , Inflamação/metabolismo , Fígado/metabolismo , Reação de Fase Aguda , Animais , Antígenos CD/metabolismo , Northern Blotting , Receptor gp130 de Citocina , Endopeptidases/metabolismo , Hepatócitos/metabolismo , Humanos , Hibridização In Situ , Interleucina-6/metabolismo , Cinética , Lipopolissacarídeos/metabolismo , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Especificidade da Espécie , Fatores de Tempo , Regulação para Cima
11.
Comp Hepatol ; 3(1): 10, 2004 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-15566573

RESUMO

BACKGROUND: The cytokine leukemia inhibitory factor (LIF) mediates its biological effects through binding to its high affinity receptor made of the low-affinity LIF receptor subunit gp190 (LIF-R) and the gp130 subunit. LIF exerts several important effects in the liver, however, data on liver expression of LIF are scarce. The aim of this study was to examine the expression of LIF and LIF-R in human liver. RESULTS: LIF expression, analyzed by immunohistochemistry, was barely detectable in normal liver but was strong within cirrhotic fibrous septa and was found in spindle-shaped cells compatible with myofibroblasts. Accordingly, cultured human liver myofibroblasts expressed high levels of LIF as shown by ELISA and Northern blot. Biological assay demonstrated that myofibroblast-derived LIF was fully active. RT-PCR showed expression of the LIF-D and M isoforms, and also of low levels of new variants of LIF-D and LIF-M resulting from deletion of exon 2 through alternative splicing. LIF receptor expression was detected mainly as a continuous sinusoidal staining that was enhanced in cirrhotic liver, suggestive of endothelial cell and/or hepatocyte labeling. Immunohistochemistry, flow cytometry and STAT-3 phosphorylation assays did not provide evidence for LIF receptor expression by myofibroblasts themselves. LIF secretion by cultured myofibroblasts was down regulated by the addition of interleukin-4. CONCLUSIONS: We show for the first time the expression of LIF in human liver myofibroblasts, as well as of two new isoforms of LIF mRNA. Expression of LIF by myofibroblasts and of its receptor by adjacent cells suggests a potential LIF paracrine loop in human liver that may play a role in the regulation of intra-hepatic inflammation.

12.
Mol Cancer Res ; 11(2): 133-9, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23233483

RESUMO

Reptin is overexpressed in most human hepatocellular carcinomas. Reptin is involved in chromatin remodeling, transcription regulation, or supramolecular complexes assembly. Its silencing leads to growth arrest and apoptosis in cultured hepatocellular carcinoma cells and stops hepatocellular carcinoma progression in xenografts. Reptin has an ATPase activity linked to Walker A and B domains. It is unclear whether every Reptin function depends on its ATPase activity. Here, we expressed Walker B ATPase-dead mutants (D299N or E300G) in hepatocellular carcinoma cells in the presence of endogenous Reptin. Then, we silenced endogenous Reptin and substituted it with siRNA-resistant wild-type (WT) or Flag-Reptin mutants. There was a significant decrease in cell growth when expressing either mutant in the presence of endogenous Reptin, revealing a dominant negative effect of the ATPase dead mutants on hepatocellular carcinoma cell growth. Substitution of endogenous Reptin by WT Flag-Reptin rescued cell growth of HuH7. On the other hand, substitution by Flag-Reptin D299N or E300G led to cell growth arrest. Similar results were seen with Hep3B cells. Reptin silencing in HuH7 cells led to an increased apoptotic cell death, which was prevented by WT Flag-Reptin but not by the D299N mutant. These data show that Reptin functions relevant for cancer are dependent on its ATPase activity, and suggest that antagonists of Reptin ATPase activity may be useful as anticancer agents.


Assuntos
Carcinoma Hepatocelular/enzimologia , Proteínas de Transporte/metabolismo , DNA Helicases/metabolismo , Neoplasias Hepáticas/enzimologia , ATPases Associadas a Diversas Atividades Celulares , Apoptose/fisiologia , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Proteínas de Transporte/biossíntese , Proteínas de Transporte/genética , Processos de Crescimento Celular/fisiologia , Linhagem Celular Tumoral , Sobrevivência Celular/fisiologia , DNA Helicases/biossíntese , DNA Helicases/genética , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Mutação , Transfecção
13.
World J Gastroenterol ; 16(2): 210-6, 2010 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-20066740

RESUMO

AIM: To study the protein C activation system in human liver myofibroblasts, and the effects of activated protein C (APC) on these cells. METHODS: Human liver myofibroblasts were obtained by outgrowth. Expression of protease activated receptor 1 (PAR-1), endothelial protein C receptor (EPCR) and thrombomodulin (TM) was analyzed by flow cytometry. Extracellular signal-regulated kinase (ERK)1/2 activation was assessed by Western blotting using anti-phospho-ERK antibodies. Collagen synthesis was studied with real-time reverse transcription-polymerase chain reaction (RT-PCR). Activation of protein C was studied by incubating liver myofibroblasts with zymogen protein C in the presence of thrombin and detecting the generation of APC with a colorimetric assay using a peptide substrate. RESULTS: Primary cultures of human liver myofibroblasts expressed EPCR on their surface, together with PAR-1 and TM. This receptor system was functional since exposure of myofibroblasts to APC induced ERK1/2 phosphorylation in a dose- and time-dependent manner. Furthermore, APC significantly upregulated the expression of collagen mRNA, as shown by real-time RT-PCR. Collagen upregulation was controlled through the ERK pathway as it was inhibited when using the mitogen-activated protein/extracellular signal-regulated kinase kinase inhibitor PD98059. Finally, using a cell-based colorimetric assay, we showed that intact myofibroblasts converted protein C into APC in the presence of thrombin. CONCLUSION: These data suggest that APC is a new modulator of liver myofibroblast activity and contributes to the pathophysiology of chronic liver diseases.


Assuntos
Fibroblastos/metabolismo , Fígado/metabolismo , Proteína C/metabolismo , Antígenos CD/metabolismo , Células Cultivadas , Colágeno/metabolismo , Receptor de Proteína C Endotelial , Fibroblastos/efeitos dos fármacos , Humanos , Proteína C/farmacologia , Receptor PAR-1/metabolismo , Receptores de Superfície Celular/metabolismo , Transdução de Sinais/fisiologia , Trombomodulina/metabolismo
14.
Am J Physiol Gastrointest Liver Physiol ; 293(1): G128-36, 2007 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-17379757

RESUMO

Several lines of data recently pointed out a role of the serine proteinase thrombin in liver fibrogenesis, but its mechanism of action is unknown. The aim of this study was to evaluate the effect of thrombin on the migration of human liver myofibroblasts. We show here that thrombin inhibits both basal migration and platelet-derived growth factor (PDGF)-BB-induced migration of myofibroblasts. By using a thrombin antagonist, a protease-activated receptor (PAR)-1 mimetic peptide, and a PAR-1 antibody, we show that this effect is dependent on the catalytic activity of thrombin and on PAR-1 activation. Thrombin's effect on basal migration was dependent on cyclooxygenase 2 (COX-2) activation because it was blocked by the COX-2 inhibitors NS-398 and nimesulide, and pharmacological studies showed that it was relayed through prostaglandin E(2) and its EP(2) receptor. On the other hand, thrombin-induced inhibition of PDGF-BB-induced migration was not dependent on COX-2. We show that thrombin inhibits PDGF-induced Akt-1 phosphorylation. This effect was consecutive to inhibition of PDGF-beta receptor activation through active dephosphorylation. Thus thrombin, through two distinct mechanisms, inhibits both basal- and PDGF-BB-induced migration of human hepatic liver myofibroblasts. The fine tuning of myofibroblast migration may be one of the mechanisms used by thrombin to regulate liver fibrogenesis.


Assuntos
Inibição de Migração Celular , Fígado/citologia , Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidores , Trombina/farmacologia , Becaplermina , Ciclo-Oxigenase 2/fisiologia , Fibroblastos/fisiologia , Humanos , Músculo Liso/citologia , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Proto-Oncogênicas c-sis , Receptor PAR-1/fisiologia
15.
J Biol Chem ; 279(7): 5200-6, 2004 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-14623891

RESUMO

The serine proteinase inhibitor tissue factor pathway inhibitor-2 (TFPI-2) inhibits the tissue factor-factor VIIa complex and thereby impairs factor Xa and subsequently thrombin generation. Here we show that thrombin itself up-regulates TFPI-2 mRNA and protein expression in human liver myofibroblasts, a cell type shown to express high levels of TFPI-2 (Neaud, V., Hisaka, T., Monvoisin, A., Bedin, C., Balabaud, C., Foster, D. C., Desmoulière, A., Kisiel, W., and Rosenbaum, J. (2000) J. Biol. Chem. 275, 35565-35569). This effect required thrombin catalytic activity, as shown by its abolition with hirudin. Although the thrombin effect could be mimicked by agonists of both protease-activated receptor (PAR)-1 and PAR-4, it was largely blocked by a PAR-1 blocking antibody. Transactivation of the epidermal growth factor (EGF) receptor has been reported as a common event in thrombin signaling. However, thrombin did not detectably transactivate the EGF receptor in liver myofibroblasts, and blocking the EGF receptor did not affect TFPI-2 induction. On the other hand, thrombin increased the expression of cyclooxygenase-2 (COX-2) mRNA via a MAPK-dependent pathway, and a specific COX-2 inhibitor abolished the effect of thrombin on TFPI-2 expression. Thus, thrombin, through PAR-1 signaling, up-regulates the synthesis of TFPI-2 via a MAPK/COX-2-dependent pathway. The up-regulation of TFPI-2 expression by thrombin could in turn down-regulate thrombin generation and contribute to limit blood coagulation.


Assuntos
Receptores ErbB/metabolismo , Glicoproteínas/biossíntese , Isoenzimas/metabolismo , Prostaglandina-Endoperóxido Sintases/metabolismo , Trombina/fisiologia , Regulação para Cima , Northern Blotting , Western Blotting , Catálise , Células Cultivadas , Ciclo-Oxigenase 2 , DNA Complementar/metabolismo , Regulação para Baixo , Inibidores Enzimáticos/farmacologia , Hirudinas/metabolismo , Humanos , Fígado/metabolismo , Sistema de Sinalização das MAP Quinases , Proteínas de Membrana , Fosforilação , RNA/metabolismo , RNA Mensageiro/metabolismo , Receptor PAR-1/metabolismo , Receptores de Trombina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Trombina/metabolismo , Fatores de Tempo , Ativação Transcricional
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA